Process Biochemistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio

Optimization studies to develop a low-cost medium for production of the

lipases of Rhizopus microsporus by solid-state fermentation and scale-up of
the process to a pilot packed-bed bioreactor
Luana Oliveira Pitola, Anelize Terezinha Jung Finklera, Glauco Silva Diasb,
⁎
Amanda Souza Machadoc, Gisella Maria Zanina, David Alexander Mitchellb, Nadia Kriegerc,
a
Departamento de Engenharia Química, Universidade Estadual de Maringá, Avenida Colombo, 5790, Maringá 87020-900, Paraná, Brazil
b
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Cx. P. 19046 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil
c
Departamento de Química, Universidade Federal do Paraná, Cx. P. 19081 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: A low-cost lipase preparation is required for enzymatic biodiesel synthesis. One possibility is to produce the
Biodiesel lipase in solid-state fermentation (SSF) and then add the fermented solids (FS) directly to the reaction medium
Lipases for biodiesel synthesis. In the current work, we scaled up the production of FS containing the lipases of Rhizopus
Rhizopus microsporus microsporus. Initial experiments in flasks led to a low-cost medium containing wheat bran and sugarcane bagasse
Solid-state fermentation
(50:50 w/w, dry basis), supplemented only with urea. We used this medium to scale-up production of FS, from
Packed-bed bioreactor
Scale-up
10 g in a laboratory column bioreactor to 15 kg in a pilot packed-bed bioreactor. This is the largest scale yet
reported for lipase production in SSF. During scale-up, the hydrolytic activity of the FS decreased 57%: from
265 U g−1 at 18 h in the laboratory bioreactor to 113 U g−1 at 20 h in the pilot bioreactor. However, the es-
terification activity decreased by only 14%: from 12.1 U g−1 to 10.4 U g−1. When the FS produced in the la-
boratory and pilot bioreactors were dried and added directly to a solvent-free reaction medium to catalyze the
esterification of oleic acid with ethanol, both gave the same ester content, 69% in 48 h.

1. Introduction
The most common process for the production of biodiesel involves
the transesterification of fats and oils with short chain alcohols, espe-
cially methanol, using a homogeneous alkaline catalyst. Biodiesel can
also be produced using lipases to catalyze either transesterification or
esterification reactions. This enzymatic route is more environmentally
friendly than the alkaline catalysis route, as it avoids the generation of
large volumes of alkaline wash water. Although biodiesel is produced
using the enzymatic route at industrial scale in a few plants in the USA
and China [1,2], the high cost of the lipases and the long reaction times
prevent the widespread adoption of this technology [3].
Traditionally, lipases have been produced by submerged fermenta-
tion (SmF) [4]. However, solid-state fermentation (SSF) represents an
interesting alternative to reduce production costs. In fact, Castilho et al.
[5] showed that the cost of producing lipases by SSF is about a third of
the cost of producing them by SmF. This is because SSF allows the
utilization of cheap agro-industrial byproducts as substrates and re-
quires a lower total capital investment [5,6].
Over the past decade, our group has been working on a strategy that
reduces the costs of producing lipases by SSF significantly in compar-
ison to the process considered by Castilho et al. [5], who included the
downstream steps of extraction and recovery. Our strategy involves
simply air-drying the fermented solids at the end of the fermentation
and then adding them directly to the reaction medium to catalyze es-
terification and transesterification reactions [7–12]. This strategy also
avoids the need for a separate immobilization step. It has recently been
adopted by other groups [13,14].
Initially, we used fermented solids to catalyze biodiesel synthesis in
media in which the substrates were dissolved in solvents such as n-
hexane and n-heptane [7]. More recently, we have used solvent-free
media [8–12]. The use of such media not only increases volumetric
productivities but also avoids the need for solvent recovery and re-
cycling. Our best results were obtained using fermented solids produced
by growing Burkholderia cepacia LTEB11 (later reclassified as Bur-
kholderia lata) on a mixture of sugarcane bagasse and sunflower seed
meal. With this solid, we achieved a conversion of 95% in 46 h for
transesterification of soybean oil with ethanol [8] and a conversion of

⁎
Corresponding author at: Departamento de Química, Universidade Federal do Paraná Cx. P. 19081 Centro Politécnico, Curitiba 81531-980, Paraná, Brazil.
E-mail address: nkrieger@ufpr.br (N. Krieger).

http://dx.doi.org/10.1016/j.procbio.2017.07.019
Received 14 April 2017; Received in revised form 16 July 2017; Accepted 24 July 2017
1359-5113/ © 2017 Elsevier Ltd. All rights reserved.

Please cite this article as: Pitol, L.O., Process Biochemistry (2017), http://dx.doi.org/10.1016/j.procbio.2017.07.019
L.O. Pitol et al. Process Biochemistry xxx (xxxx) xxx–xxx

88% in 24 h for the ethyl esterification of a mixture of free fatty acids Table 1
derived from soybean soapstock acid oil [12]. Matrix and results of the PBD used to select the components of the nutrient solution.
Our results with fermented solids containing strains of Burkholderia
Variables Units Symbols Levels used
are promising, but it would be problematic to scale up the process:
various Burkholderia strains are opportunistic pathogens and costly (−1) (+1)
containment systems would therefore be required. This led us to in-
Urea (% w/w)a X1 0.0 2.0
vestigate the potential of catalyzing biodiesel synthesis using fermented
Lactose (% w/w)a X2 0.0 2.5
solids produced with Rhizopus microsporus [10]. This strain was chosen K2HPO4 (% w/w)a X3 0.0 2.5
because varieties of this species have been used to produce the fer- MgSO4·7H2O (% w/w)a X4 0.0 0.5
mented food tempe [15]. Fermented solids produced by cultivating R. Oligoelements (% v/w)a X5 0.0 2.0
microsporus on sugarcane bagasse impregnated with the nutrient solu- Soybean oil (% w/w)a X6 0.0 20.0

tion developed by Rodriguez et al. [16] gave a conversion of 68% in

72 h for the transesterification of corn oil with ethanol in a solvent-free Experiment X1 X2 X3 X4 X5 X6 Hydrolytic Esterification
activity at activity at 18 h
system [10]. However, the fermentations were done using only 4 g of
18 h (U g−1) (U g−1)
sugarcane bagasse (dry basis) in 250-mL Erlenmeyer flasks. In fact,
although lipase production in SSF has been extensively studied at la- 1 +1 −1 +1 −1 −1 −1 114 9.2
boratory scale over the last few decades [17], there are only three re- 2 +1 +1 −1 +1 −1 −1 101 8.2
ports of lipase production involving more than 1 kg of dry substrate 3 −1 +1 +1 −1 +1 −1 34 2.4
4 +1 −1 +1 +1 −1 +1 254 16.5
[18–20]. All three of these studies involved tray bioreactors, with a 5 +1 +1 −1 +1 +1 −1 125 9.0
maximum loading of 3 kg (dry mass) per tray. 6 +1 +1 +1 −1 +1 +1 62 7.7
The aim of the current work was to demonstrate the feasibility of 7 −1 +1 +1 +1 −1 +1 35 1.9
producing fermented solids containing the lipases of R. microsporus in a 8 −1 −1 +1 +1 +1 −1 44 2.8
9 −1 −1 −1 +1 +1 +1 29 2.3
pilot packed-bed bioreactor. The work involved three steps: first, we
10 +1 −1 −1 −1 +1 +1 76 7.9
developed a nutrient solution that contains fewer components than the 11 −1 +1 −1 −1 −1 +1 30 2.3
medium used by Zago et al. [10] in order to reduce costs; second, we 12 −1 −1 −1 −1 −1 −1 36 2.8
increased the scale of the SSF from 10 g to 15 kg (dry mass); third, we
a
demonstrated that the dry fermented solids produced in the pilot Based on total dry substrate.

bioreactor can be used to esterify oleic acid with ethanol in a solvent-

free system.
2. Materials and methods
2.1. Raw materials
The reagents used were ethanol (99.5%) and urea from Synth (São
Paulo, Brazil), lactose from Nuclear (São Paulo, Brazil), K2HPO4 from
Qhemis (São Paulo, Brazil), MgSO4·7H2O, n-heptane (99.5%) and n-
hexane (99.5%) from Vetec (Rio de Janeiro, Brazil) and oleic acid
(90%) and methyl nonadecanoate (99.8%) from Sigma-Aldrich (St.
Louis, EUA). Sugarcane bagasse and wheat bran were kindly donated by
Usina de Álcool Melhoramentos (Jussara, Brazil) and Anaconda
Industrial e Agrícola de Cereais (Curitiba, Brazil), respectively.
2.2. Fungal strain and inoculum
A strain of Rhizopus, originally isolated in Guadalajara, Mexico, and
previously characterized as Rhizopus microsporus CPQBA 312-07 DRM
was used [10]. A spore suspension was prepared by growing the fungus
on potato dextrose agar at 30 °C for 7 days and harvesting the spores
with a Tween 80 solution (0.01% w/v). The spore concentration in the
suspension was determined using a Neubauer chamber.
2.3. Statistical optimization in erlenmeyer flasks
The optimization study started from the medium and culture con-
ditions proposed by Rodriguez et al. [16] and routinely used by our
group for lipase production by R. microsporus [10]. The medium, which
consists of sugarcane bagasse impregnated with a nutrient solution, is
referred to as “standard medium”. In the present work, a 50:50 (w/w)
mixture of sugarcane bagasse and wheat bran was used, rather than
only sugarcane bagasse.
The inclusion of wheat bran brings nutrients that are not present in
sugarcane bagasse. A Plackett-Burman Design (PBD) was therefore used
to select the components of the nutrient solution of Rodriguez et al.
[16] that influenced the hydrolytic and esterification activity of the
fermented solid. The components tested were (i) urea; (ii) lactose; (iii)
K2HPO4; (vi) MgSO4·7H2O; (v) oligoelement solution (containing, per
liter, 10 g EDTA, 2.0 g MnCl2·4H2O, 2.8 g CoSO4·7H2O, 1.5 g
CaCl2·2H2O, 0.2 g CuCl2·2H2O and 0.3 g ZnSO4·7H2O, pH 4.0) and (vi)
soybean oil. In this study, 12 experiments were used to evaluate the six
factors. Each factor was evaluated at levels of “ + 1” (with addition of
the component) and “-1” (without addition of the component). The
experimental design of the PBD (factors and tested range) is shown in
Table 1. The results were analyzed using Statistica 10 (Statsoft™) and
the factors with confidence levels of more than 95% (p < 0.05) were
considered to have significant effect on hydrolytic and esterification
activity and were used for further optimization using a Central Com-
posite Rotatable Design (CCRD).
In the PBD, each 250-mL flask contained 5 g (dry mass) of a mixture
of 50% sugarcane bagasse and 50% wheat bran (w/w, dry matter) and
was autoclaved (121 °C, 15 min). The nutrient solution, containing
soybean oil, urea, lactose, K2HPO4, MgSO4·7H2O, and the oligoelement
solution (described above) were autoclaved separately and then added
to the flask, to give an initial moisture content of 84% (w/w, wet basis)
and the component concentrations dictated by the statistical planning.
A spore suspension was added to the moist substrate to give 3 × 107
spores per gram of dry solid substrate [16]. The water in the spore
suspension was taken into account in the initial moisture content. The
moisture content was determined in an infrared balance (Gehaka IV
2000, São Paulo, Brazil) and reported on a wet basis. Flasks were in-
cubated at 40 °C for 18 h, conditions that have previously been shown
to give maximal lipase activities [16].
After the PBD, we conducted a Central Composite Rotatable Design
(CCRD) to evaluate the influence of the urea concentration, the initial
moisture content and the percentage of wheat bran on the hydrolytic
and esterification activity at 18 h. There were 20 experiments, corre-
sponding to a full 23 factorial design, with six additional axial points
(i.e. at coded values of +1.68 and −1.68) and six replicates of the
central point (Table 2). The results were analyzed using Statistica 10
(Statsoft™), where the effects of the variables and respective errors were
calculated at the 95% confidence level. An analysis of variance
(ANOVA) was done and the values of F from the Fisher test were used to
determine the significance of the model. The response surfaces were
obtained with the definition of the optimal operating ranges for each

2
L.O. Pitol et al. Process Biochemistry xxx (xxxx) xxx–xxx

Table 2
Matrix and results (experimental and predicted) of the CCRD used to optimize the culture medium.

Variables Units Symbols Levels used

(−1.68) (−1) (0) (+1) (+1.68)

Initial moisture content (% w/w)a X1 59.9 64.0 70.0 76.0 80.1

Urea (% w/w)b X2 1.3 3.0 5.5 8.0 9.7
Wheat bran (% w/w)b X3 39.8 48.0 60.0 72.0 80.2

Experiment X1 X2 X3 Hydrolytic activity (U g−1) Hydrolytic Activity (U g−1) Esterification activity (U g−1) Esterification activity (U g−1)
(Experimental at 18 h) (Predicted at 18 h) (Experimental at 18 h) (Predicted at 18 h)

1 −1 −1 −1 264 262 13.3 13.4

2 1 −1 −1 79 77 7.8 7.8
3 −1 1 −1 76 99 5.4 5.8
4 1 1 −1 174 158 11.5 10.6
5 −1 −1 1 156 197 10.4 11.4
6 1 −1 1 48 51 3.3 3.0
7 −1 1 1 77 105 7.5 7.8
8 1 1 1 174 202 9.9 9.8
9 −1.68 0 0 207 167 10.5 9.3
10 1.68 0 0 89 93 5.4 6.3
11 0 −1.68 0 132 120 9.2 9.0
12 0 1.68 0 135 110 8.1 8.3
13 0 0 −1.68 202 213 11.2 11.5
14 0 0 1.68 243 196 9.9 9.2
15 0 0 0 242 244 12.4 12.6
16 0 0 0 244 244 12.6 12.6
17 0 0 0 249 244 12.6 12.6
18 0 0 0 244 244 12.5 12.6
19 0 0 0 239 244 12.4 12.6
20 0 0 0 243 244 12.9 12.6

a
Wet basis.
b
Based on total dry substrate.

response variable.
In the CCRD, each 250-mL flask contained 5 g of the substrate
mixture (dry basis), with the proportions of wheat bran and sugarcane
bagasse determined by the statistical planning. The flasks were auto-
claved (121 °C, 15 min). Urea solutions were autoclaved separately
(121 °C, 15 min) and then added to the flasks, to give the initial
moisture contents (w/w, wet basis) and urea concentrations (w/w,
based on total dry substrate) dictated by the statistical planning. A
spore suspension was added to the moist substrate to give 3 × 107
spores per gram of dry solid substrate and flasks were incubated at
40 °C for 18 h [16].
After the fermentations, the whole fermented solids (which contain
lipases, residual solid substrates and fungal biomass) were lyophilized
for 24 h at −45 °C and 0.1 mbar in a Jouan LP3 Lyophilizer (Allerod,
Frederiksborg, Denmark). Lyophilized fermented solids were main-
tained at −18 °C in sealed plastic bags.
For calculation of the cost of the culture medium, the costs of the
raw materials were obtained from the database Aliceweb, which is
hosted by the Brazilian Ministry of Development, Industry and Foreign
Trade (MDIC) [21]. The average FOB prices for the period from January
to December 2016 were considered.
2.4. Solid-state fermentation in the laboratory column bioreactor
The substrate, composed of 50% wheat bran and 50% sugarcane
bagasse (w/w, dry basis), plus sufficient urea solution to obtain an in-
itial concentration of urea of 3.4% (w/w, based on total dry substrate)
and the desired initial moisture content were autoclaved separately
(121 °C, 15 min). After cooling, they were mixed together and in-
oculated with a spore suspension to give 3 × 107 spores per gram of dry
solid substrate. The moisture content after inoculation was 65% (w/w,
wet basis). SSF was performed in glass columns (4 cm internal diameter
and 21 cm height). Each column was packed with 10 g (dry mass) of the
solid culture medium and incubated in a water bath set at 40 °C. Water-
saturated air was injected into the bottom of each column at a rate of
100 cm3 min−1, which we have previously found to be suitable for
respirometry studies [22]. One column was sacrificed at each sampling
time and the fermented solids were lyophilized (see section 2.3).
The CO2 production and O2 consumption were continuously mon-
itored using infrared and electrochemical sensors, respectively
(PASPORT Carbon Dioxide Gas Sensor PS-2110 and PASPORT Oxygen
Gas Sensor PS-2126A, PASCO Scientific, California, USA). The rate of
metabolic heat production was calculated based on a factor of 520 kJ
per mol of O2 consumed [23]. The RQ (respiratory quotient) was cal-
culated by dividing the CO2 evolution rate by the O2 uptake rate [24].
2.5. Inoculum for the pilot bioreactor
Based on a preliminary study (results not shown), the protocol de-
scribed as follows was developed in order to produce sufficient spore
inoculum for the pilot bioreactor. Six stainless-steel trays
(19 cm × 26 cm × 3.5 cm) were prepared, each containing 183 g (dry
mass) of a mixture of 86% parboiled rice and 14% rice husk (w/w, dry
basis). This mixture was moistened with sufficient water (which had
been used to boil potatoes, 300 g of diced potatoes per liter of water) to
give a moisture content of 37.5% (w/w, wet basis). The moist substrate
was autoclaved (121 °C, 20 min) and a spore suspension was added to
give 1.7 × 107 spores per gram of dry solid substrate. The bed depth
was 30 mm. The trays were incubated at 30 °C for 7 days. The spores
were suspended by adding 5 mL of sterile Tween 80 solution (0.01% w/
v) per gram of dry substrate and then the suspension was filtered in a
funnel with sterile gauze to remove residual substrate. The spore con-
centration in the suspension was determined using a Neubauer
chamber.
2.6. Solid-state fermentation in the pilot packed-bed bioreactor
The pilot fermentations were undertaken in the same pilot packed-

3
L.O. Pitol et al. Process Biochemistry xxx (xxxx) xxx–xxx

Fig. 1. The pilot bioreactor. (a) Overview of the

bioreactor and the air preparation system. Key: (1)
air blower; (2) air filter; (3) humidification tower;
(4) water reservoir; (5) water pump; (6) water entry
into the humidification tower; (7) supply of satu-
rated air to bioreactor; (8) return of excess water
back to the reservoir; (9) measurement of inlet air
temperature; (10) air inlet; (11) measurements of
bed temperature; (12) air outlet; (13) measurement
of outlet air temperature; (14) data acquisition
equipment; (15) to gas washer. (b) Design details of
the pilot packed-bed bioreactor; Key: (1) 1-mm
aperture wire mesh supported on a perforated base
plate; (2) thermocouple at 5-cm height; (3) thermo-
couple at 18-cm height; (4) thermocouple at 33-cm
height; (5) thermocouple at 46-cm height; (6) elec-
tric motor with variable frequency; (c) locations
where the samples were removed at the end of the
fermentation for mapping the bed. Samples from the
bottom were collected at around 2-cm height, from
the middle at about 20-cm height and from the top at
about 40-cm height. This figure is reproduced from
Biz et al. [25] with the kind permission of Elsevier
and is a modified version of the figure originally
given in Pitol et al. [22].

Table 3
Comparison between the raw materials cost of the improved culture medium and the standard medium.

Component Price per kg of Component concentration in the Price per kg of culture Price per unit of hydrolytic Price per unit of esterification
component (US$/kg) culture medium (% w/w)a medium (dry mass) (US activity (US$/MUb) activity (US$/MUb)
$/kg)

Improved medium
Sugarcane bagasse 0.04 50 0.02 0.08 1.63
Wheat bran 0.13 50 0.07 0.27 5.69
Urea 0.23 3.2 < 0.01 < 0.01 0.06
Total 0.09 0.35 7.38

Standard medium
Sugarcane bagasse 0.04 100 0.04 0.20 2.86
Urea 0.23 2.0 < 0.01 0.02 0.33
Lactose 1.88 2.5 0.05 0.23 3.36
K2HPO4 1.46 2.5 0.04 0.18 2.61
MgSO4·7H2O 0.26 0.5 < 0.01 < 0.01 0.09
Soybean oil 0.73 20.0 0.15 0.72 10.43
Total 0.28 1.36 19.68

a
Based on total dry substrate.
b
MU means megaunits.

bed bioreactor as that used by Pitol et al. [22], Biz et al. [25] and
Finkler et al. [26]. The stainless-steel bioreactor has a bed capacity of
200 L. It has a perforated base plate (70 cm × 60 cm) that is covered by
a wire mesh with 1-mm apertures (Fig. 1a and b) [22,25,26]. Humid air
flows upwards through the base plate into the bed. In the first fer-
mentation, in order to obtain an inlet air temperature of 40 °C, the same
temperature as that used in the column bioreactor, the reservoir used to
feed the humidification column was maintained at 42 °C: Since the air
line between the humidification column and the bioreactor was not
insulated, the temperature of the air entering the bioreactor was 2 °C
lower than the temperature of the reservoir. In the second fermentation,
in an attempt to avoid the problem with condensation of water on the
inside walls of the headspace that occurred during the first fermenta-
tion, the reservoir was maintained at 36 °C, giving an inlet air
temperature of 34 °C. The air flow rate was kept constant in all fer-
mentations at 90 m3 h−1, resulting in a superficial air velocity of
0.06 m s−1.
Two fermentations were carried out with 7.5 kg of wheat bran and
7.5 kg of sugarcane bagasse (dry mass), which resulted in an initial bed
height of 40 cm. The solid substrate, sufficient solution of urea to give
an initial concentration of 3.4% (w/w, based on total dry substrate) and
the remaining water needed to obtain an initial moisture content of
65% (w/w, wet basis) were autoclaved (121 °C, 2 h). After cooling, the
solid substrate was placed in the bioreactor. The cooled water, the urea
solution and sufficient spore suspension needed to give 3 × 107 spores
per g of dry substrate were mixed together and then also added to the
bioreactor. The bioreactor was then rotated for 30 min at 5 rpm to
homogenize the bed. After this period, the bed surface was leveled

4
L.O. Pitol et al.
Fig. 2. Profiles for solid-state fermentation with Rhizopus microsporus CPQBA 312-07
DRM in the column bioreactor. This fermentation involved 6 g of wheat bran and 6 g of
sugarcane bagasse (dry basis) and a water bath temperature set at 40 °C. (a) hydrolytic
and esterification activity of the fermented solid; (b) O2 uptake rate and CO2 evolution
rate; (c) respiratory quotient. Values plotted represent the mean of duplicate col-
umns ± the standard error of the mean. If not visible, the error bars are smaller than the
symbols.
using a rake, the bioreactor was closed and the air supply system was
connected.
During the fermentation, samples were collected only from the top
of the bed. At the end of the fermentation (i.e. at 24 h), samples were
removed from five different horizontal positions (C1, C2, C3, C4 and
C5) at the bottom (2 cm height), in the middle (20 cm height) and at the
top (40 cm height) of the bed (Fig. 1c) [22].
2.7. Esterification reactions in solvent-free medium
Reactions were carried out in a solvent-free system, in 50-mL flasks
(with polytetrafluoroethylene caps) containing 45 mmol of ethanol
(2.1 g) and 30 mmol of oleic acid (8.5 g). The mixture was pre-in-
cubated for 10 min on a rotary shaker at 200 rpm and at the stated
temperature, and the reactions were started by adding 1.0 g of dry
fermented solid. The reactions were conducted at 200 rpm and 40 °C.
The ethyl-ester content of samples was determined by GC analysis using
5
Process Biochemistry xxx (xxxx) xxx–xxx
a GC-2010 (Shimadzu Co., Kyoto, Japan). The sample preparation,
column, detector and conditions were as described by Dias et al. [12].
The ester content (in mass percent) was determined relative to the peak
area of the internal standard [27].
2.8. Determination of lipase activities
The hydrolytic activity of the dry fermented solids was assessed by
the titrimetric method as described by Soares et al. [9] except that olive
oil (67 mmol L−1) was used instead of triolein and 150 mg lyophilized
fermented solids was added. One unit of activity (U) was defined as the
release of 1 μmol of fatty acid per minute, under the assay conditions.
Esterification activity was determined by direct addition of the lyo-
philized fermented solids to the reaction medium following the method
of Soares et al. [9], using 5 mL of a mixture containing 210 mmol L−1
of ethanol and 70 mmol L−1 of fatty acid in n-hexane and 500 mg of
lyophilized fermented solids. Incubation was at 200 rpm and 40 °C. Free
fatty acid contents were determined in 60-μL samples using the Lowry-
Tinsley method [28] and used to calculate ester production. One unit
(U) of esterification activity was defined as the production of 1 μmol of
ester per minute, under the assay conditions. Both the hydrolytic ac-
tivity and the esterification activity were expressed as units of activity
per gram of dry fermented solid (i.e. U g−1). Throughout this paper, the
error represents the standard error of the mean.
3. Results
3.1. Statistical optimization in erlenmeyer flasks
The effects of the six components of the nutrient solution on the
hydrolytic and esterification activities at 18 h of fermentation were
evaluated using a Plackett-Burman Design (Table 1). For both activities,
only the urea concentration (X1) had a significant effect (p < 0.05)
(Table S1, supplementary material).
The urea concentration and two other variables, namely the initial
moisture content and the percentage of wheat bran in the total dry
substrate, were optimized using a Central Composite Rotatable Design.
The matrix and the experimental and predicted results for hydrolytic
and esterification activity obtained at 18 h of fermentation are pre-
sented in Table 2.
All independent terms influenced the hydrolytic and esterification
activities significantly (p < 0.05, Table S2, supplementary material).
The models are significant at the 95% confidence limit, since the cal-
culated F values for hydrolytic activity (11.2) and esterification activity
(32.7) were higher than the tabulated F value (3.0). Additionally, the
coefficients of determination (R2) obtained for the hydrolytic and es-
terification activity models were 0.91 and 0.97, respectively, indicating
that the models fitted the experimental data well. The following
quadratic models were obtained for hydrolytic activity (H, U g−1) and
esterification activity (E, U g−1) at 18 h:
H=244.5-21.8X1-40.6X12-2.9X2-45.8X22-5.1X3-14.3X32+61.0X1X2+9.5X1X3
+17.6X2X3 (1)
E=-12.6-0.9X1-1.7X12-0.2X2-1.4X22-0.7X3-0.8X32+2.6X1X2-0.6X1X3+1.0X2X3
(2)
where X1, X2 and X3 are the coded values for the initial moisture con-
tent, the urea concentration and the percentage of wheat bran, re-
spectively. The three-dimensional response surface plots described by
these equations are given in the supplementary material (see Figs. S1
and S2).
The optimal conditions predicted by the model for hydrolytic ac-
tivity were the same as those predicted by the model for esterification
activity, namely an initial moisture content of 65% (w/w, wet basis), a
urea concentration of 3.4% (w/w, dry substrate) and a solid substrate
L.O. Pitol et al.
composed of 50% of wheat bran and 50% sugarcane bagasse. Under
these conditions, the models predicted a hydrolytic activity at 18 h of
260 U g−1 and an esterification activity at 18 h of 13.4 U g−1.
An experiment was carried out in triplicate under the optimized
conditions to validate the model. Both the hydrolytic activity of
262 ± 8 U g−1 and the esterification activity of 12.3 ± 0.5 U g−1
were very close to the values predicted by the models. This hydrolytic
activity is higher than the value of 203 ± 5 U g−1 obtained with the
standard medium. On the other hand, the esterification activity is
slightly lower than the value of 14 ± 1 U g−1 obtained with the
standard medium.
The optimization by factorial design allowed reduction of the cost of
the culture medium used to produce the fermented solid (Table 3). With
the addition of wheat bran to the substrate, it was possible to eliminate
the components of the nutrient solution used in the standard medium,
with the exception of urea. The cost decreased from US$0.28 kg−1 for
the standard medium to US$0.09 kg−1 (both on a dry mass basis) for
6
Process Biochemistry xxx (xxxx) xxx–xxx
Fig. 3. Profiles for solid-state fermentation with
Rhizopus microsporus CPQBA 312-07 DRM during the
fermentation with the air inlet set at 40 °C in the
pilot bioreactor and mapping of the bed at the end of
the fermentation. (a) hydrolytic activity and ester-
ification activity of the fermented solid at the top of
the bed; (b) moisture contents of the fermented solid
at the top of the bed; (c) temperatures at bed heights
of 5, 18 and 33 cm; (d) air temperatures at the inlet
and outlet; (e) hydrolytic activity of the fermented
solid after 24 h at different positions within the bed;
(f) esterification activity of the fermented solid after
24 h at different positions within the bed; (g)
moisture contents of the fermented solid after 24 h at
different positions within the bed. Values plotted
represent the mean of triplicate analyses ± the
standard error of the mean. If not visible, the error
bars are smaller than the symbols.
the improved medium proposed in this work, a reduction of 68%. The
price per unit of activity reduced by 74% on the basis of hydrolytic
activity and by 63% on the basis of esterification activity.
3.2. Fermentation in the laboratory column bioreactor
The study in the column bioreactor was carried out to obtain time
course data for lipase activity and respirometry. The hydrolytic and
esterification activities followed very similar profiles (Fig. 2a). After a
lag phase of about 6 h, the activities increased quickly, peaking at 18 h,
at which time the hydrolytic activity was 265 U g−1 and the ester-
ification activity was 12.1 U g−1. The decrease in these activities after
the peak was also relatively quick. The profiles for the oxygen uptake
rate (OUR) and carbon dioxide evolution rate (CER) (Fig. 2b) had
shapes that were similar to the activity profiles (Fig. 2a). The maximum
OUR was 180 mmol kg−1 h−1, at 18 h. Based on a factor of 520 J of
metabolic heat produced per mmol of O2 consumed [23], this value
L.O. Pitol et al.
corresponds to a maximum heat production rate of 26 W kg−1. The
respiratory quotient (RQ) was around 1.25 from 7 to 18 h and then
decreased, reaching a value of 1 at the end of the fermentation (Fig. 2c).
3.3. Fermentation in the pilot bioreactor
The first pilot fermentation was conducted with 7.5 kg of wheat
bran and 7.5 kg of sugarcane bagasse (dry mass), resulting in a bed
height of 40 cm. The inlet air temperature in this fermentation was set
at 40 °C.
The profiles of hydrolytic and esterification activities obtained for
the samples collected from the top of the bed (Fig. 3a) were similar to
those obtained in the column bioreactor (Fig. 2a), with a rapid increase
in lipase activity between 6 and 18 h, followed by a decrease after 18 h.
However, the maximum values of activities obtained at 18 h in this
pilot fermentation were lower, 83 U g−1 for hydrolytic activity and
7
Process Biochemistry xxx (xxxx) xxx–xxx
Fig. 4. Profiles for solid-state fermentation with
Rhizopus microsporus CPQBA 312-07 DRM during the
fermentation with the air inlet set at 34 °C in the
pilot bioreactor and mapping of the bed at the end of
the fermentation. (a) hydrolytic activity and ester-
ification activity of the fermented solid at the top of
the bed; (b) moisture contents of the fermented solid
at the top of the bed; (c) temperatures at bed heights
of 5, 18 and 33 cm; (d) air temperatures at the inlet
and outlet; (e) hydrolytic activity of the fermented
solid after 24 h at different positions within the bed;
(f) esterification activity of the fermented solid after
24 h at different positions within the bed; (g)
moisture contents of the fermented solid after 24 h at
different positions within the bed. Values plotted
represent the mean of triplicate analyses ± the
standard error of the mean. If not visible, the error
bars are smaller than the symbols.
7.5 U g−1 for esterification activity.
There was an increase in the moisture content at the top of the bed
during the period of 14–18 h (Fig. 3b). This occurred because the room
temperature was not controlled and during this period it reached a
value 10 °C lower than the air temperature in the headspace of the
bioreactor. Since the bioreactor walls were not insulated, moisture
condensed on the inside walls of the headspace and dripped onto the
top of the substrate bed, leaving it visibly wetter than normal.
The low metabolic heat production by R. microsporus and the use of
50% sugarcane bagasse, which prevented bed compaction, resulted in a
negligible axial temperature gradient in the bed (Fig. 3c). Throughout
the fermentation, the temperature in the bed remained within 1 °C of
the temperature of the inlet air (Fig. 3d).
At the end of the fermentation, samples were removed from various
heights and horizontal positions in the bed to characterize the homo-
geneity. The hydrolytic activity varied between 34 and 72 U g−1
L.O. Pitol et al. Process Biochemistry xxx (xxxx) xxx–xxx

Mass of dry solid substrate and bed depth (type of activity, percentage of

3 kg, bed depth not informed (hydrolytic activity, 126%, 96 h)

the activity obtained in flasks and time for which reported)

15 kg, 40 cm (esterification activity, 85%, 20 h)

15 kg, 40 cm (hydrolytic activity, 43%, 20 h)
1 kg, 1.5 cm (hydrolytic activity, 84%, 72 h)

1 kg, 1.5 cm (hydrolytic activity, 83%, 72 h)

Scaled-up bioreactor
Fig. 5. Profiles for the esterification of oleic acid with ethanol in a solvent-free system
obtained using the fermented solids produced in the column bioreactor and in the pilot
bioreactor. Reaction conditions: 1 g of dry fermented solids, 2.1 g of ethanol and 8.5 g of
oleic acid (i.e. molar ratio of ethanol to oleic acid of 1.5:1), with incubation at 40 °C and
200 rpm. Values plotted represent the mean of duplicate reactions ± the standard error
of the mean. If not visible, the error bars are smaller than the symbols.

Mass of dry solid substrate (type of activity, activitya,

, 120 h, olive oil)

10 g (hydrolytic activity, 384 U g−1, 72 h, olive oil)

, 96 h, olive oil)

5 g (hydrolytic activity, 262 U g−1, 18 h, olive oil)

5 g (esterification activity, 12.3 U g−1, 18 h, oleic
(Fig. 3e) and the esterification activity varied between 4.2 and
7.1 U g−1 (Fig. 3f); both had a slight tendency to be higher in the
middle of the bed. In the case of the moisture content, all values fell

time for which reported and substrate)

−1

−1
within the range of 61–74% (w/w, wet basis), with a tendency for the

10 g (hydrolytic activity, 629 U g

10 g (hydrolytic activity, 791 U g

moisture content to be lower in the middle of the bed (Fig. 3g).
Given the increase in moisture content observed at the top of the
bed in the first pilot fermentation, the second pilot fermentation was
undertaken with the same substrate loading, but with the inlet air
Erlenmeyer Flasks

temperature set at 34 °C. During this fermentation, the room tempera-

ture was, at most, 4 °C lower than the air temperature in the headspace
of the bioreactor. As a result, the problem of condensation of water on
the inside walls of the headspace did not occur and the moisture con-

acid)
tent at the top of the bed did not increase significantly.
The maximum hydrolytic activity obtained for the samples collected
from the top of the bed in this pilot fermentation was 113 U g−1 at 20 h
(Fig. 4a), which is greater than the value of 83 U g−1 obtained at 18 h
Packed-bed
bioreactor

in the first pilot fermentation (Fig. 3a). The esterification activity also
Type of

increased, from 7.5 U g−1 at 18 h in the first pilot fermentation

Tray

Tray

Tray

(Fig. 3a) to 10.4 U g−1 at 20 h in the second (Fig. 4a). Although the
maximum hydrolytic activity obtained in the second pilot fermentation
Microorganism and solid substrate (% by mass on dry basis)

Aspergillus niger MTCC 2594, wheat bran (75%) and gingelly

Aspergillus niger MTCC 2594, wheat bran (62.5%), coconut

Aspergillus tamari, wheat bran (75%) and gingelly oil cake

was 43% of the value of 265 U g−1 obtained in the laboratory column
Rhizopus microsporus, wheat bran (50%) and sugarcane

bioreactor, the esterification activity was 86% of the value of

12.1 U g−1 obtained in the laboratory fermentation. This suggests that
bagasse (50%) supplemented with urea (3.4%)

the solid produced in the pilot fermentation has the potential to be used
(25%) supplemented with gingelly oil (0.5%)

in the synthesis of esters.

oil cake (25%) and wheat rawa (12.5%)

As with the first fermentation, the bed temperature within the

Scale-up studies of lipase production by solid-state fermentation.

bioreactor was well controlled. There was no bed compaction and the
temperature in the bed remained within 1 °C of the temperature of the
inlet air (Fig. 4b and c).
At the end of the fermentation, the uniformity of the lipase activity
was different from that obtained in the first fermentation. In this fer-
mentation, there was no trend with height, although there was a greater
Activities are per gram of dry substrate.
oil cake (25%)

variation within each horizontal plane (Fig. 4d and e): The sample
standard deviations for the hydrolytic activity of the five samples re-
moved from the same horizontal plane ranged from 2 to 8 U g−1 in the
first pilot fermentation and from 10 to 19 U g−1 in the second. Like-
wise, the sample standard deviations for the esterification activity in
Dayanandan et al. [20]
Edwinoliver et al. [19]

the same horizontal plane ranged from 0.7 to 0.9 U g−1 in the first
fermentation and from 0.9 to 1.4 U g−1 in the second. The moisture
Mala et al. [18]

contents in this fermentation were more uniform, varying from 61% to

This work

68% (w/w, wet basis).

Reference
Table 4

8
L.O. Pitol et al.
Fig. 6. Relationship between the esterification and hydrolytic activity in the laboratory-
column fermentation and the two pilot fermentations. For the laboratory column bior-
eactor, the data are from Fig. 2a. For the first fermentation in the pilot bioreactor, which
was done with an inlet air temperature of 40 °C, the data are from Fig. 3a. For the second
fermentation in the pilot bioreactor, which was done with an inlet air temperature of
34 °C, the data are from Fig. 4a.
3.4. Esterification reactions in solvent-free medium
The esterification activities reported above for the studies under-
taken in the laboratory column bioreactor and pilot packed-bed bior-
eactor were measured using a standardized method involving the sub-
strates dissolved in n-hexane [9]. An experiment was done to confirm
the ability of these fermented solids to catalyze the esterification re-
action in a solvent-free medium. The fermented solids used in this ex-
periment had the same esterification activity, 10.9 U g−1, but different
hydrolytic activities, 240 U g−1 for the fermented solid produced in the
laboratory fermentation and 120 U g−1 for the fermented solid pro-
duced in the pilot fermentation. Conversions of 69% in 48 h were ob-
tained in this solvent-free system with both fermented solids (Fig. 5).
4. Discussion
The current work describes the scale-up of an SSF process for the
production of fermented solids containing the lipases of R. microsporus
and shows that these solids can be used to catalyze the esterification of
fatty acids in a solvent-free system. The scale was increased from 10 g
of a 50:50 mixture (w/w, dry basis) of wheat bran and sugarcane ba-
gasse in a laboratory column bioreactor to 15 kg (dry basis) of the same
substrate in a pilot packed-bed bioreactor. This corresponds to ap-
proximately 40 kg of moist substrate and is the largest scale at which
lipase production by SSF has been reported to date.
The three previous studies of the scale-up of lipase production by
SSF used tray bioreactors with substrate loadings of less than 3 kg of dry
substrate per tray (Table 4) [18–20]. At large scale, tray bioreactors
have disadvantages compared to the packed-bed bioreactor used in the
current work. In a tray bioreactor, the air flows around the bed; since it
is not forced to flow through the void spaces of the bed, O2 and heat
transfer within the bed are quite limited. If high temperatures or the
lack of O2 within the bed are to be avoided, then the bed height must be
limited to a few centimeters. In fact, the results of both Mala et al. [18]
and Edwinoliver et al. [19] show evidence of heat transfer limitations in
their trays: even with bed heights of only 1.5 cm, bed temperatures
4–8 °C above the temperature of the surrounding air were measured
[18,19]. This contrasts with the current work, in which the bed tem-
perature never reached values more than 1 °C higher than the tem-
perature of the inlet air, even with a 40-cm high bed.
9
Process Biochemistry xxx (xxxx) xxx–xxx
The three previous studies did not present an explicit strategy for
scale up of the process to commercial scale. However, since all three
studies involved trays, the implicit scale-up rule would be to build a
larger reactor with more trays. The disadvantage is that tray processes
are difficult to automate and therefore tend to be labor intensive.
Additionally, the design of tray chambers has received little attention.
For example, there is no quantitative information about how much
space to leave between trays in order to maximize volumetric pro-
ductivity (i.e., the rate of product formation per unit volume of tray
chamber) while avoiding problems with heat removal from the trays
[29]. In the case of the current work, which involves packed beds, the
strategy for scale up to commercial scale would be that suggested by
Pitol et al. [22], namely to maintain the bed height used at pilot scale
and to increase the capacity by increasing the width of the bed to
several meters, while maintaining the superficial air velocity at a con-
stant value. Pitol et al. [22] suggested maintaining a bed height of
40 cm in an attempt to avoid high temperatures at the top of the bed
and bed compaction. In the current work, these problems were not
observed and it might be possible to increase the bed height beyond
40 cm in such a scale-up procedure. This would allow a bioreactor with
a smaller footprint per tonne of substrate, although further work must
be done to confirm this.
The good performance observed in the SSF process studied in the
current work can be attributed to both the substrate and the micro-
organism: The maximum metabolic heat production obtained for R.
microsporus in the column bioreactor was 26 W kg−1 and the substrate
contained 50% sugarcane bagasse. This combination is similar to that
used by Biz et al. [25] for the production of pectinases in the same pilot
bioreactor; they used A. oryzae, which gave a maximum metabolic heat
production rate of 21 W kg−1 in the column bioreactor, and a substrate
consisting of 48.4% sugarcane bagasse. As in the current work, bed
compaction did not occur and the temperature in the bed was well
controlled throughout their fermentation [25]. Part of this good per-
formance can be attributed to the fact that the high content of su-
garcane bagasse (50%) in the substrate ensured a high porosity within
the bed, completely avoiding the problems of bed compaction that Pitol
et al. [22] experienced when producing pectinases with a substrate
composed of 90% wheat bran and only 10% sugarcane bagasse. These
compaction problems resulted in the formation of preferential flow
paths and a consequent generalized overheating of the bed [22]. The
other part of the good performance can be attributed to the relatively
low rate of metabolic heat production: Pitol et al. [22] used a strain of
A. niger that gave a much higher maximum heat production rate of
87 W kg−1 in the column bioreactor and, upon scaling up to the pilot
bioreactor, they obtained temperature gradients in the bed of up to 5 °C,
even at times at which preferential flow paths had not yet developed.
The cost of the improved medium is about 30% of that of the
standard medium. Although the esterification activity of the fermented
solid produced using the improved medium was 14% lower than that
obtained with the standard medium, the greater degree of reduction in
raw material costs gives a lower cost per unit of activity: 7.38 US$/MU
for the improved medium against 19.68 US$/MU for the standard
medium. Crucially, the preparation of the improved medium does not
involve the steps of washing, drying and sieving of the sugarcane ba-
gasse that are used in the preparation of the standard medium, making
the improved medium cheaper still.
The elimination of downstream steps for enzyme recovery also
contributes to the economic viability of our process, especially when
compared to the production of lipases by submerged fermentation. For
example, production of lipase AK, a Pseudomonas fluorescens lipase that
Amano Pharmaceuticals produces by submerged fermentation, requires
multiple downstream processing steps, namely filtration, ultrafiltration,
microfiltration, precipitation, centrifugation, vacuum drying, crushing
and sieving [30]. We avoided the costs of such steps by adding the
lyophilized fermented solid directly to the reaction medium to catalyze
the esterification reaction. Importantly, although we dried the
L.O. Pitol et al.
fermented solids by lyophilization, which is a relatively expensive
process, we have recently shown that lipase activity is maintained if the
solids are dried by passing dry air at room temperature through the bed
at the end of the fermentation [9].
In the present study, the hydrolytic activity fell from 265 U g−1 to
113 U g−1 as the scale increased from 10 g (column bioreactor) to 15 kg
(pilot bioreactor), a decrease of 57%. On the other hand, the ester-
ification activity fell from 12.1 U g−1 to 10.4 U g−1, a decrease of only
14%. The difference in the behavior of hydrolytic activity and ester-
ification activity with increase in scale may be due to differences in the
production of different lipase isoforms. This possibility is supported by
a plot of the esterification activity against the hydrolytic activity for the
laboratory column fermentation and the two pilot fermentations
(Fig. 6). This plot shows that the ratio of hydrolytic activity to ester-
ification activity remains slightly over 20 for the laboratory-scale fer-
mentation but only around 10 for both pilot-scale fermentations. In
fact, R. microsporus produces at least five different intracellular and
extracellular lipases, with the hydrolytic activity of the extracellular
forms being higher than that of the corresponding intracellular forms,
while the intracellular forms appear to be mainly responsible for the
synthetic activity [31]. This difference between intracellular and ex-
tracellular lipases has been demonstrated for the lipases of a related
species, R. oryzae, for which the methanolysis is correlated with the
amount of intracellular lipase [32]. Of course, in order to confirm our
hypothesis, it would be necessary to quantify the different isoforms
produced at the different scales and their individual hydrolytic and
esterification activities.
Our results confirm that the hydrolytic activity of the fermented
solid is not a reliable indicator of its potential for ester synthesis: the
hydrolytic activity of the fermented solid produced in the pilot fer-
mentation (120 U g−1) was only half that of the fermented solid pro-
duced in the laboratory column bioreactor (240 U g−1), but both fer-
mented solids gave the same conversion for the esterification of oleic
acid in the solvent-free system, 69% in 48 h. Although the percentage
conversions obtained in this work were lower than those obtained by
our group for ester synthesis catalyzed by fermented solids produced by
B. cepacia [8,9,12], they are better than the value of 68% in 72 h ob-
tained previously for the fermented solid produced by R. microsporus
[10]. Since R. microsporus is safer to work with than B. cepacia, our
results are promising, although further improvement in the percentage
conversion will be necessary in order for the biodiesel to meet the re-
levant specifications.
5. Conclusions
We produced a low-cost fermented solid containing lipases of
Rhizopus microsporus in a pilot packed-bed bioreactor containing 15 kg
of dry substrate, which is the largest substrate loading yet reported in
the literature for lipase production by solid-state fermentation. This
fermented solid was able to catalyze esterification of oleic acid with
ethanol in a solvent-free system, with a conversion of 69% in 48 h.
Although further improvements in the conversion are required, this
fermented solid has the potential to lower the costs of a large-scale
processes for lipase-catalyzed biodiesel production.
Acknowledgements
This research was supported by a “Universal” grant from CNPq
(Conselho Nacional de Desenvolvimento Científico e Tecnológico), a
Brazilian government agency for the advancement of science and
technology. Research scholarships were granted to Amanda Souza
Machado, Gisella Maria Zanin, David Alexander Mitchell and Nadia
Krieger by CNPq, to Luana Oliveira Pitol by Fundação Araucária, an
agency for the support of science and technology in the State of Paraná,
and to Anelize Terezinha Jung Finkler and Glauco Silva Dias by CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), a
10
Process Biochemistry xxx (xxxx) xxx–xxx
Brazilian government agency for the development of personnel in
higher education.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.procbio.2017.07.019.
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